| Summary: | The adenosine triphosphate-hydrolysing activities of Escherichia coli NRC 482 were investigated. These activities
could be accounted for by two enzymes, one of which was
5'-nucleotidase and the other was a Ca²⁺ -or Mg²⁺ -activated ATP phosphohydrolase (ATPase). The 5'-nucleotidase and the
Ca²⁺ -or Mg²⁺ -activated ATPase could be readily distinguished
on the basis of their properties and could be separated
from each other by ion exchange chromatography. Both soluble
and membrane-bound Ca²⁺ -or Mg²⁺ -activated ATPase activities were present in cell extracts. The membrane-bound ATPase, though firmly attached to the membrane, could be solubilized under conditions in which the membranes were depleted of divalent metal ions. This solubilized ATPase was identical to the ATPase found In the soluble fraction.
The Ca²⁺-or Mg²⁺activated ATPase was partially purified after solubilization from E. coli membranes by gel filtration and ion exchange chromatography. The ATPase activity was shown to correspond to a single protein band after polyacrylamide gel electrophoresis. The enzyme had a molecular weight of 375,000. It was activated by divalent but not by monovalent cations. Optimal activity occured at pH 9.5 and at ion to substrate ratios of 2:5 and 2:3 with Mg²⁺ and Ca²⁺, respectively. ADP inhibited the ATPase reaction. The ATPase was stable at 22°- 2k° but was rapidly inactivated at 4°, Glycerol stabilized the enzyme at both
temperatures. In these, and in other respects, the Ca²⁺
or Mg²⁺ -activated ATPase of E. coli resembled the ATPase
activities of other bacterial strains, which in turn have
a resemblance to the mitochondrial and chloroplast ATPases.
To help establish the function of the Ca²⁺ -or Mg²⁺ -activated ATPase the levels of the enzyme were measured in E. coll grown under a variety of conditions. The level of the enzyme in the cell was not affected by the carbon source, phase of growth, aeration of the culture or the extent of catabolite repression, although these are conditions under which the efficiency of oxidative phosphorylation
is known to vary in vivo. These findings can be reconciled with the current hypothesis regarding the function of the ATPase in E. coli. === Medicine, Faculty of === Biochemistry and Molecular Biology, Department of === Graduate
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